Audiogenic kindling activates expression of vasopressin in the hypothalamus of Krushinsky-Molodkina rats genetically prone to reflex epilepsy.

The present study analysed the effects of audiogenic kindling on the functional state of the vasopressinergic system of Krushinsky-Molodkina (KM) rats. KM rats represent a genetic model of audiogenic reflex epilepsy. Multiple audiogenic seizures in KM rats lead to the involvement of the limbic structures and neocortex in the epileptic network. The phenomenon of epileptic activity that overspreads from the brain stem to the forebrain is called audiogenic kindling and represents a model of limbic epilepsy. In the present study, audiogenic kindling was induced by 25 repetitive audiogenic seizures (AGS) with 1 AGS per day. A proportion of KM rats did not express AGS to sound stimuli, and these rats were characterised as the AGS-resistant group. The data demonstrated that audiogenic kindling did not change activity of extracellular signal-regulated kinase 1/2 or cAMP response element-binding protein, although it led to an increase in vasopressin (VP) expression in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN). Additionally, we observed a decrease in GABAergic innervation of the hypothalamic neuroendocrine neurones after audiogenic kindling, whereas glutamatergic innervation of the SON and PVN was not altered. By contrast, analysis of AGS-resistant KM rats did not reveal any changes in the activity of the VP-ergic system, confirming that the activation of VP expression was caused by repetitive AGS expression, rather than by repetitive acoustic stress. Thus, we suggest that overspread of epileptiform activity in the brain is the main factor that affects VP expression in the hypothalamic magnocellular neurones.

Effects of ERK1/2 kinases inactivation on the nigrostriatal system of Krushinsky-Molodkina rats genetically prone to audiogenic seizures.

Recently, we demonstrated that inhibition of ERK1/2 activity by SL-327 treatment blocks seizure behavior in Krushinsky-Molodkina (KM) rats, which was mediated by altering of GABA and glutamate release mechanism in the hippocampus. Basal ganglia representing various subcortical cell groups play a significant role in the regulation of motor activity, including epileptiform seizures. OBJECTIVES:  To verify if nigrostriatal system could be also affected by SL-327 treatment we analyzed the expression of tyrosine hydroxylase, D1 and D2 dopamine receptors, NR2B subunit of NMDA receptor as well as vesicular glutamate transporter VGLUT2 and glutamic acid decarboxylases GAD65/67 in the striatum and substantia nigra of KM rats. METHODS: Animals were injected i.p. with SL-327 (50 mg/kg) 60 min before audio stimulation. After audiogenic stimulation the brains of control and SL 327 treated rats were removed for further immunohistochemical and biochemical analysis. RESULTS:  Obtained results demonstrated a decrease activity in synapsin I, and accumulation of VGLUT2 in the striatum after blockade of audiogenic seizure (AGS) by SL 327 that could lead to inhibition of glutamate release. While in the striatum GAD65/67 level was diminished, in the substantia nigra GAD65/67 was increased showing enhanced inhibitory output to the compact part of the substantia nigra. Analysis of dopaminergic system showed a significant reduction of tyrosine hydroxylase activity and expression in the substantia nigra, and decreased D1 and D2 receptor expression in the striatum. In summary, we propose that changes in the nigrostriatal system could be mediated by inhibitory effect of SL 327 on AGS expression.

Inhibition of ERK1/2 signaling prevents epileptiform behavior in rats prone to audiogenic seizures.

It has recently been proposed that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. We hypothesized that inhibition of ERK1/2 activity could prevent audiogenic seizures by altering GABA and glutamate release mechanisms. Krushinsky-Molodkina rats, genetically prone to audiogenic seizure, were recruited in the experiments. Animals were i.p. injected with an inhibitor of ERK1/2 SL 327 at different doses 60 min before audio stimulation. We demonstrated for the first time that inhibition of ERK1/2 activity by SL 327 injections prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. The obtained data also demonstrated unchanged levels of GABA production, and an increase in the level of vesicular glutamate transporter 2. The study of exocytosis protein expression showed that SL 327 treatment leads to downregulation of vesicle-associated membrane protein 2 and synapsin I, and accumulation of synaptosomal-associated protein 25 (SNAP-25). The obtained data indicate that the inhibition of ERK1/2 blocks seizure behavior presumably by altering the exocytosis machinery, and identifies ERK1/2 as a potential target for the development of new strategies for seizure treatment. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. Here we report that inhibition of ERK1/2 by SL 327 prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. Accumulation of VGLUT2 was associated with differential changing of synaptic proteins VAMP2, SNAP-25 and synapsin I. The obtained data indicate that the inhibition of ERK1/2 alters neurotransmitter release by changing the exocytosis machinery, thus preventing seizures.

Effects of selective Bcl-2 inhibitor HA14-1 treatments on functional activity of magnocellular vasopressinergic neurons of rat hypothalamus.

In this study we examined whether in vivo treatments with Bcl-2 inhibitor HA14-1 can affect the function of vasopressinergic system of rat. HA14-1 is a novel organic compound that has micromolar affinity for Bcl-2 and Bcl-xL and acts as a mimetic of BH3-only proteins by antagonizing the anti-apoptotic Bcl-2 proteins and triggering Bax-dependent apoptosis. We found that intrahypothalamic injections of HA14-1 did not induce apoptosis of vasopressin (VP) cells of supraoptic nucleus, but led to activation of VP synthesis and release, resulting in decreased diuresis. Our data has also demonstrated that injections of HA14-1 increased phospho-MEK1/2, phospho-CREB and phospho-Elk-1 levels in magnocellular neurons. Thus we propose that injections of HA14-1 into the hypothalamus do not lead to neuronal death, but change the functional activity of VP neurons of hypothalamus centres.

Apoptotic signaling proteins: possible participation in the regulation of vasopressin and catecholamines biosynthesis in the hypothalamus.

The role of apoptotic signaling proteins for long-lived neurons in the mature brain is poorly understood. Recently, we have shown that water deprivation leads to the activation of vasopressin (VP) secretion and expression of Bcl-2 and caspase-9 apototic proteins in the hypothalamus of the rat brain. In the present work, we continued to study a possible relationship between the functional activity of neurosecretory cells of the hypothalamus and apoptosis related proteins. We found that water deprivation leads to simultaneous activation of synthesis of VP and p53 and Bcl-2 apoptotic proteins in the mouse brain. To study a possible effect of apoptotic proteins on the functional state of hypothalamic neurons, the VP and tyrosine hydroxylase (TH) synthesis were analyzed in p53, p21(Waf1/Cip1) and Bcl-2 deficient mice. Loss of p53 and Bcl-2 significantly reduced VP synthesis in paraventricular and supraoptic nuclei and TH expression in arcuat, periventricular and zona incerta nuclei of the hypothalamus. Surprisingly, in contrast with the loss of p53, the inactivation of p21(Waf1/Cip1) up-regulates the expression of VP and TH. These data indicate that p53, p21(Waf1/Cip1) and Bcl-2 proteins, besides affecting cell cycle, tumor suppression and apoptosis, may act as modulators of neurosecretory activity of hypothalamic neurons; however, this problem remains to be determined more detailed.